US10265345B2 - Use of extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for the treatment of acute cerebrovascular disease - Google Patents

Use of extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for the treatment of acute cerebrovascular disease Download PDF

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US10265345B2
US10265345B2 US13/128,254 US200913128254A US10265345B2 US 10265345 B2 US10265345 B2 US 10265345B2 US 200913128254 A US200913128254 A US 200913128254A US 10265345 B2 US10265345 B2 US 10265345B2
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adjusting
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Mansang Lau
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Vanworld Pharmaceutical Rugao Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to the treatment of acute cerebrovascular diseases.
  • it relates to extracts from rabbit skin inflamed by vaccinia virus in the manufacture of a medicament for treating acute cerebrovascular diseases.
  • Stroke one of the acute cerebrovascular diseases, is the third leading cause of death in worldwide population and induces a highest disabling rate among various diseases.
  • the incidence of cerebrovascular diseases in China ranges from about 0.12% to 0.18%, which is the second cause among population death.
  • the cerebrovascular diseases could severely affect the life quality of the elderly, bring an enormous burden to patients' family and the society. It also trends to increase in young population.
  • the cerebrovascular diseases are primarily classified into two types, hemorrhagic and ischemic, of which the latter is 60-70%, and is the most common type of cerebrovascular diseases. It is important to study the pathophysiological mechanism of ischemic cerebrovascular diseases and search for drugs which function as neuroprotection.
  • drugs currently used to clinically treat cerebral ischemia mainly comprise calcium ion antagonists (nimodipine), oxygen radical scavengers (VitE, SOD), neurotrophic factors (nerve growth factor, neurotrophic factor), excitatory amino acid antagonists, antioxidants and drugs which improve late-onset neuronal injury.
  • the extracts from rabbit skin inflamed by vaccinia virus refers to the active substances extracted from the rabbit skin inflamed by vaccinia virus, as described in Chinese patent NO. ZL98103220.6, the entirety of which is incorporated herein by reference.
  • Such extracts from rabbit skin inflamed by vaccinia virus are commercial available, with trade name of analgecine, which is manufactured by Vanworld Pharmaceutical (Rugao) Co. Ltd.
  • analgesic effects include: (1) analgesic effects, including obvious analgesic effects on hyperalgesia complexly induced by repeated cold stresses, which are achieved by activating the descending inhibition system of central nervous system; (2) effects on sense of coldness and abnormal perception: it has been showed by experiments in vivo and in vitro that such agent has the effects of changing the neuron sporadic activity of hypothalamus, suggesting that the agent has reparative and regulating effects on abnormal sense neuron sporadic activity, which is considered to be the cause for neuralgia and abnormal perception; (3) effects of improvement of peripheral blood circulation; (4) effects on regulation of autonomic nerves: it has been suggested by the experiment in vivo and in vitro that the agent can improve the symptoms of autonomic nerve system dysregulation by regulating the activity of central autonomic nerve; (5) effects on anti-allergic reaction: it has been suggested by animal experiments that the agent has effects on anti-allergic reaction type I, which has inhibitory effects on respiratory tract hypersecretion resulted from excitation
  • analgecine has a beneficial effect on cerebral ischemia in the experimental animal models.
  • the present invention provides a method for treating acute cerebrovascular diseases in mammals comprising administrating to a subject in need of thereof such extracts from rabbit skin inflamed by vaccinia virus.
  • the present invention provides the use of the extracts from rabbit skin inflamed by vaccinia virus for the manufacture of a medicament for treating acute cerebrovascular diseases in a mammal.
  • the acute ischemic cerebrovascular diseases as described herein are acute ischemic cerebrovascular diseases.
  • Cerebrovascular diseases are neural function injuries caused by abnormal blood supply of regional brain. In most countries, cerebrovascular diseases, the top three causes of all deaths, can result in a brain injury in adult. Cerebrovascular disease is a major cause for endangering the health of the middle-aged and the aged people, and a major cause of death or disability of the middle-aged and the aged people in most countries. Ischemic cerebrovascular diseases mostly resulted from cerebrovascular occlusion, which is generally known as embolism or thrombosis; and its pathophysiology changes are highly complicated.
  • the acute ischemic cerebrovascular diseases as described herein include, but not limited to cerebral embolism, transient cerebral ischemia attack, cerebral thrombosis, cerebral arteriosclerosis, cerebral arteritis, steal syndrome of cerebral artery, cranial venous sinus and venous thrombus.
  • Ischemic cerebrovascular disease is caused by transient or permanent reduction of blood flow in certain areas of artery blood supply due to embolism, and its pathological processes relate to complicated temporal and spatial cascade reaction.
  • the pathophysiologic mechanism of cerebral ischemia has been widely investigated in recent years.
  • the direct reason of this disease is that each artery has its basic ranges of blood supply in the brain, and the occlusion of the artery will lead to softening of the brain tissues in the respective areas, resulting in the corresponding clinical syndromes, in which the neurological symptomatologic injuries (such as contralateral limb hemiplegia) caused by middle cerebral artery occlusions are the most common.
  • the neurological symptomatologic injuries such as contralateral limb hemiplegia
  • middle cerebral artery obstructions are the most common.
  • the percentage of the middle cerebral artery obstructions is large, so the pathological processes simulated by an animal model of middle cerebral artery obstruction (MCAO) have great similarity to that of clinical strokes.
  • MCAO
  • analgecine can remarkably improve the neurological symptoms of animals. Therefore, in one embodiment, analgecine can be used for the treatment of cerebrovascular diseases by improving the neural function.
  • analgecine is used for treatment of cerebrovascular diseases by reducing the areas of cerebral infarction.
  • Brain is the most active organ in metabolism with the least energy and oxygen storage per se.
  • the consumed oxygen of the brain tissues accounted for 20% of total body oxygen consumption under the resting state.
  • Neurons constitute the primary parts which consume oxygen in the cerebral cortex or whole brain, and are highly sensitive to ischemia and hypoxia injury. When there is no source of fresh oxygen, the tissues can only consume their high energy phosphate compound storage, and obtain the energy by means of metabolising the stored glucoses and glycogens into MDA.
  • analgecine is used for treatment of cerebrovascular diseases by decreasing the level of lactic acid in brain tissues.
  • SOD is an important antioxydant enzyme which can inhibit free radical reactions effectively, and high SOD activities represent strong antioxydant abilities.
  • the SOD activities of the brain tissues in rats decreased significantly and accordingly the abilities of free radicals elimination decreased after cerebral ischemia injuries.
  • the results showed that the SOD activities can be enhanced via intervention of analgecine, indicating that analgecine may play a role in neuroprotection by increasing the antioxidant abilities of brain tissues. Accordingly, in one embodiment, analgecine is used for treatment of cerebrovascular diseases by increasing the SOD activities.
  • analgecine can have a protective effect on nerve cell injury.
  • H 2 O 2 is an important reactive oxygen component which is involved in the onset of nervous system diseases such as cerebral ischemia, trauma, brain aging, Alzheimer's disease etc. It will peroxidate membrane lipid, decrease cell membrane fluidity, change components and activities of intracellular proteins, make chromatin concentrated and DNA broken, and finally result in cell death. Therefore, in one embodiment, analgecine is used to improve H 2 O 2 -induced injury of PC12 cell.
  • Excitatory amino acids such as glutamic acid
  • Glutamic acid can damage nerve cell line and primary nerve cell in dose dependent manner. It is responsible for the increased intracellular calcium ion and the blocked cystine uptake, and it induces the loss of intracellular reduced glutathione (GSH), the increased oxygen radical and nerve cell death. Therefore, H 2 O 2 or glutamic acid-induced nerve cell injury model can be used as a screening model of neuroprotective agents.
  • analgecine is useful to improve glutamic acid-induced injury of PC12 cell, inhibit the expression or excretion of ICAM-1 in endothelial cell of the brain vessels, and/or inhibit T- and B-lymphocyte transformations.
  • T-lymphocytes exhibits increased cell volume, robust metabolism, increased synthesis of protein and nucleic acid, and be able to achieve lymphoblast divisions after stimulated by specific antigen or nonspecific mitogen during culturing in vitro.
  • the level of lymphocyte transformation rate reflects the immunologic function of cells in individuals. Therefore, lymphocyte transformation test is widely used for determining one of the indicators of immunologic functions of the cells in an individual, and also for screening immunomodulators.
  • the experiment studies have found that analgecine has certain inhibitory effects on lymphocyte transformation. Therefore, in one embodiment, analgecine is useful to inhibit the transformations of T- and B-lymphocytes.
  • the inner membranes of vessels which are made of endothelium consisted of endothelial cells, play an important role in maintaining vessel homeostasis. Functions of endothelial cells can be easily affected by ingredients in the blood because such cells are in contact with the blood directly.
  • the endothelial cells are activated under the pathological conditions, such as hypoxia, chronic and acute inflammation, ischemia injury, and in turn express some adhesion molecules: ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), E-selectin and P-selectin.
  • Adhesion molecules play an important role in pathological processes of blood vessel endothelium and vessel, wherein ICAM-1 plays a key role in the close adhesion of leukocyte to endothelium. Therefore, in one embodiment, analgecine is useful to inhibit endothelial cells in cerebral vessels to express or excrete ICAM-1.
  • the present invention relates to an analgesic and its manufacturing method.
  • the analgesic is produced by preparing an active agent from pox skin tissue of rabbit and mixing the active agent with a pharmaceutically acceptable excipient, wherein the active agent is prepared by the steps of inoculating rabbit with vaccinia virus, extraction in solvent, acid treatment, alkaline treatment, absorption, rinsing, concentration and so forth.
  • An analgesic is a class of drug that acts primarily on the central nervous system and selectively inhibits pain without affecting the sensation.
  • Opioid alkaloids and their synthetic substitutes are the typical centrally-acting analgesics. They have strong analgesic effect, but their side effects are very serious and repeated dosage can cause addiction, tolerance, and respiratory depression. Hitherto, there has been no clinically potent analgesic with minimal side effects.
  • the present invention is to provide an analgesic with strong analgesic effect, and minimal side effects and a method for manufacturing such analgesic.
  • the analgesic of the present invention contains an active agent and a pharmaceutically acceptable excipient, wherein said active agent contain aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, urocanic acid, uracil, hypoxanthine, xanthine, and thymine.
  • active agent contain aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, urocanic acid, uracil, hypoxanthine, xanthine, and thymine.
  • the substances mentioned above ranging in ( ⁇ g/ml) are: 0.2-0.5 aspartic acid, 0.1-0.4 threonine, 0.3-0.8 serine, 0.7-1.4 glutamic acid, 0.3-0.7 glycine, 0.4-0.9 alanine, 0.1-0.4 valine, 0.1-0.3 isoleucine, 0.1-0.4 leucine, 0.2-0.6 tyrosine, 0.1-0.4 phenylalanine, 0.05-0.2 lysine, 0.1-0.4 histidine, 12.0-22.5 urocanic acid, 6.5-12.1 uracil, 0.7-1.4 hypoxanthine, 5.4-10.2 xanthine, and 1.3-2.5 thymine.
  • This preparation is a colorless or pale yellow liquid with pH value of 7.0-8.0, UV absorption at 265-275 nm, positive in ninhydrin reaction, negative in test for various proteins, positive in 3,5-dihydroxy toluene-hydrochloric acid reaction, and positive in arsenomolybdate color reaction.
  • the active agent is prepared by the steps of:
  • the active agent is prepared by the steps of:
  • the analgesic of the present invention is obtained by mixing the active agent with a pharmaceutically acceptable excipient.
  • the analgesic can be used in different clinical application with various pharmaceutical forms such as injection, tablet, ointment, capsule, granule, and so forth. It is preferable to use injection form.
  • the pharmaceutically acceptable excipient includes various pharmaceutically acceptable carriers.
  • the excipient can be distilled water for injection, normal saline, vegetable oil for injection, glucose injection, propylene glycol, polyethylene glycol, and so forth.
  • the excipient can also be various stabilizers, emulsifiers and so forth.
  • the excipient can be adjuvant such as starch, lactose, mannitol and so forth; binding agent such as crystalline cellulose, acacia, corn starch, gelatin, polyethylene, polyvinyl alcohol, polyvinyl pyrrolidone and so forth; disintegrant such as carboxymethyl cellulose, polyethylene glycol, potato starch; lubricant such as talcum powder, magnesium stearate and so forth; wetting agent such as glycerol and so forth.
  • the excipient can be fatty oil, paraffin, lanolin, petrolatum, ethylene glycol, glycerin and so forth as substrate.
  • analgesic of the present invention has analgesic effect on various diseases.
  • the diseases include various neuralgia, low back pain, biliary colic, angina, arterial embolization pain, severe pain caused by various reasons such as trauma and burns, intra-operative and post-operative pain, pain due to peptic ulcer diseases, dysmenorrhea, pain due to uterine contractions after childbirth, headache, pain caused by cancer or tumor, and so forth.
  • the analgesic of the present invention can improve immune functions. Additionally, the analgesic of the present invention has minimal side effects.
  • the analgesic of the present invention was tested on 20 patients with cutaneous pruritus.
  • the analgesic of the present invention was injected subcutaneously daily with 1 ampule (3 ml), at least 3 times a week, for 2 weeks.
  • 70% of the subjects had complete or moderate improvement while 95% of tested subjects had mild or better improvement.
  • muscle and subcutaneous injection of the analgesic of the present invention is effective and safety for pain relief.
  • the study using immune related inflammation as indicator with consideration of 48 hours autologous PCA inhibition activity, and anti-complement activity acting on macrophages showed that the analgesic of the present invention could effectively promote the activation of macrophages, significantly inhibit the IgE antibody production and the related 48-hr autologous PCA reaction in mouse model of type I allergic reaction, and suppress anti-complement activity in type II allergic response model.
  • the relationship between effect and dosage was linear. Therefore, the analgesic of the present invention can suppress the inflammation related to the immune function and, in turn, improve immune functions.
  • the acute toxicity of the analgesic of the present invention was tested in mouse and rat model with a single oral or intraperitoneal or subcutaneous administration.
  • the dosage for intraperitoneal and subcutaneous administration was 6,000 unit/kg while the dosage for oral administration is 10,000 unit/kg.
  • the rats were intraperitoneally administrated with the analgesic of the present invention at dosages of 30, 60, 120 unit/kg for 28 days. No death was observed.
  • the examinations of urine, eye, hematology, histopathology, and anatomy showed that the analgesic of the present invention did not cause any pathological changes. Therefore, the analgesic of the present invention has very low toxicity.
  • the antigenic activity experiment in guinea pigs and mice showed that the analgesic of the present invention had no antigenic activity.
  • total number of injury of the gross and histopathological observation was counted as safety test indicator. The test showed that the analgesic of the present invention had very low local toxicity.
  • the subcutaneous injection of the analgesic of the present invention at 3 dosages was applied to the mothers to test the effect on the animals, fetus, and the neo-natal.
  • the results showed that the drug did not affect the maternal health status, body weight, and amount of in-take during gestation and lactation.
  • the lactation and anatomical observation of the mother was found no abnormality related to the substance injected. There was no abnormality found in the appearance, skeletal system, and different organs of the fetus due to the injected test substance. It was also observed that the injected substance did not affect the survival rate, body weight changes, appearance changes, various functions during weaning, behavior, and reproduction function of neo-natal and the off-spring.
  • the recovery test using bacteria showed that the analgesic of the present invention did not cause a genetic mutation observed.
  • the chromosomal abnormality test of mammalian cell culture (CHL) showed that the analgesic of the present invention did not cause chromosome aberrations.
  • An in vitro cytogenetic assay for micronuclei cytotoxicity was conducted on ICR (Crj:CD-1) mice and showed that no chromosome aberration was observed.
  • FIG. 1 Effects of analgecine on the volume of cerebral infarction 48 hours after permanent MCAO
  • mice Male Wistar rats, weighed 280-300 g, were provided by Beijing Vitalriver Experimental Animal Center (License: SCXKjing 2007-0004). The animals were housed in conventional manner under room temperature at 23-25° C. before or after surgery, and food and water provided ad libitum.
  • the rats were randomized into 6 groups: sham group, injury model group (vehicle control), analgecine dosing group (10 u/kg, 20 u/kg, 40 u/kg), edaravone dosing group (3 mg/kg).
  • the drugs were administrated to the animals 5 times starting 2 hrs after surgery (2 h, 6 h, 20 h, 24 h, 47 h). The animals were sacrificed 48 hours after surgery, and then each test was conducted.
  • String inserting method was used for preparing a cerebral ischemia model with reference to the method of occlusion via string ligation for rat middle cerebral artery established by Zea Longa et al [5-6] .
  • Marks were made at the starting point and 18.5 mm away from the starting point of string, which was washed with 75% (v/v) ethanol, and placed in heparinized saline at 1:2500 until use.
  • Rats were intraperitoneally injected with 10% chloral hydrate solution at 400 mg/kg.
  • Rats were fixed in dorsal position, and made an incision on skin just at the middle of the neck.
  • the left common carotid artery (CCA) was exposed after layers of tissues bluntly dissected.
  • ICA Internal carotid artery
  • ECA external carotid artery
  • a bulldog clamp was used for clamping at the proximal end of CCA, and “V” type incision about 2 mm in diameter was made between the ligation of ECA and the bifurcate point.
  • the nylon string was gently inserted into CCA from the incision, and then was passed through the bifurcate point between internal carotid artery and external carotid artery into the internal carotid artery.
  • the nylon string was slowly pushed towards the part of ICA in the intracranial direction for about 18.5 ⁇ 0.5 mm in depth until slight resisting force appeared, and then the other end of nylon string was passed through the beginning of the MCA to reach a thinner anterior cerebral artery.
  • the blood flow blockage in left middle cerebral artery has been achieved at this moment, then the ICA was sutured to secure the nylon string and to avoid bleeding, followed by suturing in layers with 1 cm of the end of the nylon string left outside the skins.
  • the anesthesia before surgery and vascular separation operation were only conducted in the sham group without ligating and introducing the String. Room Temperature was Maintained at 24-25° C. Throughout the Surgery Process.
  • the particular scoring method is as follows:
  • the rats were decapitated after being graded.
  • the brain tissues were removed and placed in a freezer at ⁇ 20° C. for 10 min, then at room temperature. After Olfactory bulb, cerebellum and lower brain stem were removed, 4 coronal incisions were made into 5 consecutive brain coronal sections at interval of 2 mm as shown in FIG. 1 .
  • the first incision was made at the middle of connection line between procerebral pole and chiasma opticum; the second was at chiasma opticum; the third was at the infundibular stalk site; and the forth was between infundibular stalk and caudate nucleus.
  • the brain sections were quickly immersed in 5 ml TTC solution (containing 1.5 ml 4% TTC solution+3.4 ml distilled water+0.1 ml 1 mol/L K 2 HPO 4 solution) on bath at 37° C. in the dark for 30 min.
  • the sections were turned over once every 7-8 min Normal brain tissues were in rose color after staining, while infarction tissues were white and were clearly defined.
  • the brain sections of each group were arranged in order, and the images were taken and saved. Image analysis system software was used for process and statistical analysis.
  • the volume of cerebral infarction was determined by the sum of the products of the area of each brain section for each animal and 2 mm, the thickness of each section.
  • Infarction volume was expressed as percentage of the volume of hemisphere in order to remove the errors caused by cerebral edema.
  • volume of cerebral infarction (%) (volume of contralateral hemisphere in surgery ⁇ volume of contralateral hemisphere in surgery without infarction)/volume of contralateral hemisphere in surgery ⁇ 100% 4.
  • the brains were removed after the rats were decapitated. The left hemisphere was separated from the right, and 1 mm frontal pole and 1 mm occipital pole were removed.
  • the brain tissues were placed in cold homogenization buffer (Tris-HCl 50 mmol/L, NaCl 150 mmol/L, CaCl 5 mmol/L, PMSF 0.1 mmol/L, pH 7.4) at volume ratio of 1:10, then minced to small pieces, and homogenized at 4° C. The concentration of the protein was determined by Bradford's method [10] .
  • the substrate lactic acid
  • lactate dehydrogenase was catalyzed into pyruvic acid by lactate dehydrogenase in the presence of oxidized coenzyme I at pH 10, then the resulting pyruvic acid could react with 2,4-dinitrophenylhydrazine to give brownish dinitrophenylhydrazone pyruvate.
  • the content of pyruvic acid can be determined by colorimetric assay, from which the activities of lactate dehydrogenase may be derived.
  • lactate dehydrogenase The activities of lactate dehydrogenase were determined according to the instruction in the kit. 10 ⁇ l homogenate and 10 ⁇ l 5 g ⁇ L ⁇ 1 coenzyme I were added to the buffered medium solution and incubated at 37° C. for 15 min; then 50 ⁇ l 0.2 g ⁇ L ⁇ 1 2,4-dinitrophenylhydrazine was added, incubated at 37° C. for 15 min; 150 ⁇ l 0.4 mol/L NaOH was added and mixed, then the absorbance was read at 440 nm after calibration. The standard curve was plotted with sodium pyruvate standards.
  • Superoxide anion radicals which were produced by xanthine and xanthine oxidase reaction system, can oxidize hydroxylamine to form nitrite which will be developed into purplish red by the chromogenic agent.
  • the SOD in the sample specifically inhibits superoxide anion radicals, and accordingly the produced nitrite will be reduced.
  • Anaesthetized rats have recovered their consciousness and have developed various degrees of focal neural dysfunction after cerebral ischemia, representing as lacking strength of lower left limbs, turning left when walking upright, tumbling to the left and even not being able to walk, and even showing disorder of consciousness; when grabbed by tail, exhibiting flexion of the left forelimb, retraction, as well as extension of the hind limb and turning right.
  • the animals in model group exhibited obvious symptoms of neural injury after cerebral ischemia with significant increase in score of neural function (P ⁇ 0.01); 40 u/kg analgecine improved the neural function symptoms significantly (P ⁇ 0.05), whereas 10, 20 u/kg groups had no significant effects of improvement. There was no significant difference between the edaravone group and model group. The results are shown in Table 1.
  • the level of lactic acid in the brain tissues of rats increased to 0.98 ⁇ 0.09 mmol/g protein after ischemia injury with significant difference (P ⁇ 0.01) compared to sham group; the level of lactic acid in the group of 40 u/kg analgecine decreased significantly to 0.70 ⁇ 0.07 mmol/g protein with statistical significance (P ⁇ 0.05) compared to model group; the level of lactic acid in edaravone group decreased significantly to 0.64 ⁇ 0.08 mmol/g protein with statistical significance (P ⁇ 0.05) compared to model group.
  • the results are shown in Table 2.
  • the level of SOD in brain tissues of rats decreased to 165.84 ⁇ 13.14 nmol/g protein with significant difference (P ⁇ 0.01) compared to sham group after ischemia injury; the level significantly increased in 20 u/kg and 40 u/kg analgecine dosing groups compared to model group (P ⁇ 0.05); the level of SOD in edaravone group significantly increased compared to model group (P ⁇ 0.01).
  • the results are shown in Table 3.
  • PC12 cells were purchased from Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsase, polylysine, standard fetal bovine serum (FBS), 1640 medium, LDH kit (available as above). All other conventional reagents are analytically pure reagents commercially available in China.
  • PC12 cells provided by Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences were cultured in complete 1640 medium (containing 10% equine serum, 5% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin) at 37° C. and 5% CO2 in a thermostatic incubator with the medium changed every 2-3 days [6] .
  • Normal control group PC12 cells were normally cultured in serum-containing DMEM medium
  • H2O2 model group the original media were removed after PC12 cell cultures were confluented into a monolayer, serum free media containing H2O2 at the final concentration of 200 ⁇ mol/L were added, and the cultures were incubated in a thermostatic incubator at 37° C. and 5% CO2 for 24 hr
  • Sample treatment group After PC12 cell cultures were confluented into monolayer, the original media were removed, a sample was added to pretreat for 1 hr, followed by H2O2 at a final concentration of 200 ⁇ mol/L, then the cultures were incubated in serum-free for 24 hr.
  • PC12 cell viability decreased to 71.94 ⁇ 3.54% which exhibited significant difference (P ⁇ 0.01) compared to normal control group after hydrogen peroxide injury; and the viability in 0.25, 0.5, 1 u/ml analgecine dosing groups significantly increased compared to model group (P ⁇ 0.05).
  • PC12 cells were purchased from Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsase, polylysine were purchased from Sigma. Standard fetal bovine serum (FBS), 1640 medium were purchased from Gibco. All other conventional reagents are analytically pure reagents commercially available in China.
  • PC12 cells were cultured in complete 1640 medium (containing 10% equine serum, 5% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin) in a thermostatic incubator at 37° C. and 5% CO2 with the medium changed every 2-3 days.
  • complete 1640 medium containing 10% equine serum, 5% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin
  • ICAM-1 ELISA assay kits were purchased from Wuhan Boster Bio-engineering Ltd. Co. Endothelial cell growth fator was provided by Roche. Fetal bovine serum was the product of Gibco. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsase, polylysine and lipopolysaccharide (LPS) were purchased from Sigma. Standard fetal bovine serum (FBS), 1640 medium were purchased from Gibco.
  • the endothelial cells of cerebral vessel were cultured in 96-well plate. LPS (10 ug/ml) was added to the wells for stimulation for 24 hrs when the endothelial cells were confluented into monolayer, then ICAM-1 was determined in the cell supernatant. The level of ICAM-1 for each sample was derived from a standard curve.
  • ICAM-1 expressed or excreted by the endothelial cells in cerebral vessels exhibited significant difference (P ⁇ 0.01) compared to the normal control group; the ICAM-1 in 0.25, 0.5, 1 u/ml analgecine dosing groups increased significantly compared to model group (P ⁇ 0.01).
  • 1640 medium (containing 10% calf serum, double-antibody, glutamine), double distilled water, saline, ConA and PMA were all purchased from Sigma.
  • mice were purchased from Institute of Zoology of Chinese Academy of Medical Sciences.
  • the spleens of Balb/C mice were collected using conventional method under aseptic condition, and were gently comminuted with a pestle followed by passing through a 200 mesh steel sieve, then washed with 1640 medium once, and centrifuged at 2000 rpm ⁇ 5 min.
  • the pellets of cells were washed with double distilled water and saline to disrupt erythrocytes, centrifuged, and the supernatant was discarded.
  • the concentration of cells were adjusted to 2 ⁇ 10 6 /ml with 1640 medium, then were placed in 96-well plates at 100 ⁇ l/well while ConA (final concentration of 5 ug/ml) or PMA (final concentration of 5 ug/ml) were added at 90 ⁇ l/well.
  • samples or cyclosporin A (final concentration of 50 nM, positive control) were added at 10 ⁇ l/well, with wells set up as blank control well (free of the inducer and the drug) or negative control well (inclusion of the inducer, but free of the drug), then incubated at 37° C. and 5% CO 2 for 72 h.
  • Lymphocyte transformation % (OD value of sample well ⁇ OD value of negative control well)/(OD value of negative control well ⁇ OD value of blank control well) ⁇ 100%
  • a strong healthy adult Japanese white rabbit weighted 2.5 kg with 3 cm test is was used as a male rabbit for antigen subculture.
  • the scrotum was disinfected with swab soaked with 70% alcohol.
  • the antigen vaccinia virus (vaccinum variolae) Lister (dried cowpox vaccinia virus, produced by the Japanese National Institute of Health Prevention (Tokyo Shinagawa Kamiosaki 2-10-35)) was defrosted and shaken well.
  • 0.2 ml antigen was taken by 1 ml syringe and injected to the central inner part of the testis.
  • the rabbit injected with the antigen was placed back to the specific animal cage which was provided with sufficient drinking water and fodder and observed twice a day.
  • testis became hardened, swollen, and purple.
  • the scrotum was cleaned with swab soaked with 70% alcohol. The scrotum was dissected and the connective tissue is removed from the testis.
  • the cut-out testis was washed once with normal saline (0.9%) and twice with PBS( ⁇ ) (80 g sodium chloride, 2 g potassium chloride, 11.5 g sodium dihydrogen phosphate, 2 g potassium dihydrogen phosphate dehydrate, 10 L injectable water).
  • PBS( ⁇ ) 80 g sodium chloride, 2 g potassium chloride, 11.5 g sodium dihydrogen phosphate, 2 g potassium dihydrogen phosphate dehydrate, 10 L injectable water.
  • the rinsed testis was blot-dried, weighed, placed in a designated container with ice. Ultimately, the testis was stored in the ⁇ 80° C.
  • the homogenate mixed with Eagles's medium at 1:1 ratio, which medium comprises 9.4 g Eagle's Powder, 12.5-22 ml 10% sodium bicarbonate, 10 ml glutamine, 1 L injectable water.
  • the mixture was aliquoted and placed in ⁇ 80° C. ultra-low temperature freezer to freeze for 1 hour.
  • the mixture was taken out and thawed at 37° C. water bath.
  • the freeze and thaw were repeated 3 times.
  • the mixture was centrifuged at low temperature (4° C., 3500 rpm, 20 minutes).
  • the mixture was aliquoted into 10 ml/tube and stored in ⁇ 80° ultra-low temperature refrigerator.
  • Antigen was taken out from the ⁇ 80° ultra low temperature freezer and placed in 30° incubator to thaw slowly. 5 ml antigen (10 ⁇ 7-10 ⁇ 8 virus/ml) obtained from Example 6 was taken using a 10 ml syringe and mixed with 500 ml PBS( ⁇ ) well to get the antigen for injection. The back of a healthy mature rabbit (2.75 kg) was shaved and then rubbed with swab soaked with 75% alcohol. The antigen prepared above was used for intradermal injection to the back of the rabbit from left to right. Each injection should be separated by 1.5 cm. Eleven injections were applied from proximal to distal. 5 columns were made on each side. Each injection contains 0.2 ml antigen.
  • the solution was filtered under low pressure with filter paper No. 5 and 0.45 ⁇ m membrane to obtain Solution C. Flushed the Solution C with nitrogen for 3 minutes to make it form foam. Then 1M hydrochloric acid was used to adjust the pH of the solution to 4.5. Then the solution was flushed with nitrogen for 3 minutes to make it form foam. 40 g activated charcoal was added and stirred continuously at 30° C. for 4 hours. After stopped stirring, let the solution set still for 30 minutes and took the supernatant. The supernatant was filtered under nitrogen-filled environment with filter paper No. 5. Then the activated charcoal was soaked with injectable water (pH 8.0) and rinsed. Filtered under nitrogen-filled environment with filter paper No. 5.
  • the Solution E was taken to the reduced pressure distiller and flushed the distiller with nitrogen.
  • the solution was distilled at 60° C. until the volume reached 5 ml and filtered with filter paper No. 5 and 0.2 ⁇ m membrane to obtain 5 ml active agent.
  • the active agent contained aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, urocanic acid, uracil, hypoxanthine, xanthine, and thymine.
  • the ranges of various substances stated above are: 0.3 aspartic acid, 0.2 threonine, 0.5 serine, 1.1 glutamic acid, 0.5 glycine, 0.6 alanine, 0.3 valine, 0.1 isoleucine, 0.3 leucine, 0.4 tyrosine, 0.2 phenylalanine, 0.1 lysine, 0.2 histidine, 18.2 urocanic acid, 9.5 uracil, 1.1 hypoxanthine, 8.6 xanthine, and 2.0 thymine.
  • the agent is colorless liquid with pH about 7.5, UV absorption at 265-275 nm, positive in ninhydrin reaction, negative in tests for various proteins, positive in 3,5-dihydroxy toluene-hydrochloric acid reaction and positive in arsenomolybdate color reaction.
  • Antigen was taken out from the ⁇ 80° ultra low temperature freezer and placed in 30° incubator to thaw slowly. 5 ml antigen (10 ⁇ 7-10 ⁇ 8 virus/ml) obtained from Example 6 was taken using a 10 ml syringe and mixed with 500 ml PBS( ⁇ ) well to get the antigen for injection. The back of a healthy mature rabbit (2.5 kg) was shaved and rubbed with swab soaked with 75% alcohol. The antigen prepared above was used for intradermal injection to the back of the rabbit from left to right. Each injection should be separated by 1.5 cm. Ten injections were applied from proximal to distal. 5 columns were made on each side. Each injection is 0.2 ml antigen.
  • the solution was filtered under low pressure with filter paper No. 5 and 0.45 ⁇ m membrane to obtain Solution C.
  • the Solution C was flushed with nitrogen for 3 minutes to make it form foam.
  • 1M hydrochloric acid was used to adjust the pH of the solution to 4.0.
  • the solution was flushed with nitrogen for 3 minutes to make it form foam.
  • 30 g activated charcoal was added and stirred continuously at 25° C. for 4 hours. After stopped stirring, let the solution set still for 30 minutes and took the supernatant.
  • the supernatant was filtered under nitrogen-filled environment with filter paper No. 5.
  • the activated charcoal was soaked with injectable water (pH 8.0) and rinsed. Filtered under nitrogen-filled environment with filter paper No. 5.
  • the Solution E was taken to the reduced pressure distiller and flushed the distiller with nitrogen.
  • the solution was distilled at 70° C. until the volume reached 5 ml. Filtered with filter paper No. 5 and 0.2 ⁇ m membrane to obtain 2 ml active agent.
  • the active agent contained aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, urocanic acid, uracil, hypoxanthine, xanthine, and thymine.
  • the agent is colorless liquid with pH about 7.0, UV absorption at 265-275 nm, positive in ninhydrin reaction, negative in test for various proteins, positive in 3,5-dihydroxy toluene-hydrochloric acid reaction and positive in arseno molybdate color reaction.
  • Antigen was taken out from the ⁇ 80° ultra low temperature freezer and placed in 30° incubator to thaw slowly. 5 ml antigen (10 ⁇ 7-10 ⁇ 8 virus/ml) obtained from Example 6 was taken using a 10 ml syringe and mixed with 500 ml PBS( ⁇ ) well to get the antigen for injection. The back of a healthy mature rabbit (3 kg) was shaved and rubbed with swab soaked with 75% alcohol. The antigen prepared above was used for intradermal injection to the back of the rabbit from left to right. Each injection should be separated by 1.5 cm. Twelve injections were applied from proximal to distal. 5 columns were made on each side. Each injection is 0.2 ml antigen.
  • the solution was filtered under low pressure with filter paper No. 5 and 0.45 ⁇ m membrane to obtain Solution C.
  • the Solution C was flushed with nitrogen for 3 minutes to make it form foam.
  • 1M hydrochloric acid was used to adjust the pH of the solution to 4.8.
  • the solution is flushed with nitrogen for 3 minutes to make it form foam.
  • 44 g activated charcoal was added and stirred continuously at 35° C. for 4 hours. After stopped stirring, let the solution set still for 30 minutes and took the supernatant.
  • the supernatant was filtered under nitrogen-filled environment with filter paper No. 5.
  • the activated charcoal was soaked with injectable water (pH 8.0) and rinsed. Filtered under nitrogen-filled environment with filter paper No. 5.
  • the Solution E was taken to the reduced pressure distiller and flushed the distiller with nitrogen.
  • the solution was distilled at 55° C. until the volume reached 4 ml. Filtered with filter paper No. 5 and 0.2 ⁇ m membrane to obtain 4 ml active agent.
  • the active agent contained aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, urocanic acid, uracil, hypoxanthine, xanthine, and thymine.
  • the ranges of various substances stated above are: 0.5 aspartic acid, 0.1 threonine, 0.8 serine, 0.7 glutamic acid, 0.3 glycine, 0.9 alanine, 0.4 valine, 0.1 isoleucine, 0.1 leucine, 0.2 tyrosine, 0.4 phenylalanine, 0.2 lysine, 0.1 histidine, 22.5 urocanic acid, 6.5 uracil, 0.7 hypoxanthine, 10.2 xanthine, and 2.5 thymine.
  • the agent is colorless liquid with pH about 8.0, UV absorption at 265-275 nm, positive in ninhydrin reaction, negative in test for various proteins, positive in 3,5-dihydroxy toluene-hydrochloric acid reaction and positive in arsenomolybdate color reaction.
  • An injection was prepared using the following formulation and the conventional method for injection preparation:
  • An injection was prepared using the following formulation and the conventional method for injection preparation:
  • An injection was prepared using the following formulation and the conventional method for injection preparation:
  • a tablet was prepared using the following formulation and the conventional method for tablet preparation:

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EP3592369A4 (en) 2017-03-06 2020-09-09 Jun Liu MEANS OF INHIBITING OR MITIGATING BETA-INDUCED DAMAGE
CN109504649B (zh) * 2017-09-15 2022-06-14 天津小西生物医药科技有限公司 使用兔皮提取物促进细胞增殖的方法
KR20200103647A (ko) * 2017-12-28 2020-09-02 가꼬우호우징 효고 이카다이가쿠 리포칼린형 프로스타글란딘 d2 합성 효소 산생 촉진제
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EP3957314A4 (en) 2019-04-17 2022-12-07 Nexus Bio-Drug Development Limited USE OF AN EXTRACT FROM VACCINIA VIRUS INFLAMMATED RABBIT SKIN TO TREAT HEMATOPOIETIC SYSTEM DAMAGE
JP7488588B2 (ja) * 2019-06-14 2024-05-22 俊熙有限公司 ワクシニアウイルスによって炎症を起こしたウサギ皮膚由来の抽出物のがん治療における使用
CN110693914A (zh) * 2019-10-28 2020-01-17 威世药业（如皋）有限公司 痘苗病毒致炎兔皮提取物在治疗类风湿性关节中的应用
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JP2012508192A (ja) 2012-04-05
JP2017052788A (ja) 2017-03-16
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